Gag gift for age 50

Diabetic retinopathy, which is classically defined as a microvasculopathy, is being viewed as a neurodegenerative disease of the retina (1). Much evidence suggests (2-8) that changes in the functional molecules and viability of neurons in the retina occur immediately after the onset of diabetes, preceding the vascular complications in both humans and experimental animals. The most prominent changes in the electroretinogram seen in the early stages of diabetes involve oscillatory potentials that are thought to derive from dopaminergic amacrine cells (9). Therefore, alterations in the dopaminergic system are thought to be among the first significant events in the development of diabetic retinopathy (2-4).

Neurotrophins are expressed and have pleiotropic effects in the nervous system (10). Among them, brain-derived neurotrophic factor (BDNF) is expressed in retinal ganglion cells (RGCs) and Muller glia in the retina (11) and is important for the survival of RGCs (12). It has also been reported (13,14) to prevent amacrine cell death. In addition to being a survival factor, BDNF acts as a synaptic modulator and has been shown (15) to cause hypertrophy of the retinal dopaminergic system in the rat retina. Although many studies have described the important roles of BDNF in the physiology and pathophysiology of the retina, there have been no reports of the involvement of neurotrophins in diabetic retinopathy. Here, we report the degeneration of dopaminergic amacrine cells accompanied by a reduction in BDNF levels in the retina of rats with streptozotocin (STZ)-induced diabetes. Furthermore, we will demonstrate the therapeutic potential of BDNF for treating neurodegeneration of dopaminergic amacrine cells in the diabetic rat retina.

RESEARCH DESIGN AND METHODS

All experimental procedures using animals were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and our institutional guidelines for the care and use of laboratory animals. Adult male Wistar rats (Japan SLC, Hamamatsu, Japan), 9 weeks of age (250-300 g), were housed in standard lighting conditions (12-h light and 12-h dark cycle) with food and water ad libitum for at least 7 days before experimentation. STZ (70 mg/kg in 50 mmol/l sodium citrate buffer, pH 4.5; Sigma, St. Louis, MO) was administered intraperitoneally under general anesthesia with diethylether inhalation (16). Nondiabetic animals were injected with an equal volume of citrate buffer. Body weight and blood glucose concentrations were measured before the injection and weekly thereafter. Diabetes was confirmed by assaying the glucose concentration in blood obtained from the tail vein using FreeStyle (Nipro, Osaka, Japan). Rats with glucose levels >250 mg/dl were classified as diabetic. Animals were anesthetized by intraperitoneal injection of an overdose of chloral hydrate and killed by decapitation 4 weeks after the STZ injection, near the end of the light phase. The retinas were processed for further analyses.

Intraocular administration of BDNF. Multiple intraocular injections, for a total of five times, were given every 3 days, beginning 2 weeks after the intraperitoneal injection of STZ or citrate buffer, under general anesthesia with diethylether and topical anesthesia with a drop of 2% lidocaine applied to the eyes. BDNF (5 p.g) in 5 [miscro]l of 0.1% BSA (Fraction V; Sigma) in balanced salt solution (BSS) (Alcon, Fort Worth, TX) was injected into the vitreous space of one eye chosen at random according to a previous report (17). An equal volume of vehicle (0.1% BSA in BSS) was injected into the other eye as a control. Retinas were dissected 2 days after the last intraocular injection. Any animal with lens damage or visible vitreous hemorrhage was excluded from the analyses.

Western blotting. To determine tyrosine hydroxylase (TH) protein levels, retinal tissues were lysed, and their protein samples were subjected to SDS-PAGE followed by Western blotting as previously reported (18). Mouse anti-TH monoclonal antibody (19) (1:10,000 dilution; a gift from Dr. Hatanaka) and peroxidase-conjugated goat anti-mouse IgG antibody (1:10,000 dilution; Jackson ImmtmoResearch Laboratories, West Grove, PA) were used as primary and secondary antibodies, respectively. Protein levels of TrkB, a high-affinity receptor for BDNF, in the retina were determined by Western blotting using mouse anti-TrkB monoclonal antibody (20) (1:3,000 dilution). Anti-[beta]-actin (1:5,000 dilution; Chemicon, Temecula, CA) was used as a loading control for Western blotting. The intensity of the bands was quantified by densitometry using NIH Image version 1.60 (National Institutes of Health, Bethesda, MD). Protein concentration was assessed by a BioRad Protein Assay Kit (BioRad Laboratories, Hercules, CA).

Enzyme-linked immunosorbent assay. Protein levels for BDNF (21), neurotrophin-3 (NT-3) (22), mid Thy-1 (23) in the retina were quantified by enzyme-linked immunosorbent assay (ELISA) as previously described. ELISA samples (in duplicate) and standards (in triplicate) were applied to titer plates. The anti-BDNF antibody used in ELISA has no cross-reactivity against other neurotrophins or growth factors and did not detect BDNF protein in samples obtained from BDNF knockout mice (21). A standard curve of recombinant BDNF (1-100 pg/well), NT-3 (1-100 pg/well), or Thy-1 (300-6,000 pg/well) was plotted for each plate. The average value of the sample was normalized against the total protein concentration.